Wang Tao, Han Chao, Tian Peng, Li Peng-Fei, Ma Xin-Long
Department of Orthopaedics, Tianjin Medical University General Hospital, Tianjin, China.
Department of Orthopaedics, Tianjin Hospital, Tianjin, China.
Orthop Surg. 2018 May;10(2):152-159. doi: 10.1111/os.12369. Epub 2018 May 10.
To determine the signaling pathways mediated by teriparatide in MLO-Y4 cell lines based on the evaluation of reactive oxygen species (ROS) through AKT pathways, which regulate apoptosis of bone cells.
We performed the DCFH-DA assay to investigate the role of ROS in MLO-Y4 cells caused by dexamethasone (Dex). Four groups were included: Dex group, Dex+NAC, Dex+ teriparatide group and control group (without any dispose). Real-time reverse transcriptase polymerase chain reaction was used to test the SOD2 and Cat mRNA expression. Western blot (WB) was used to investigate the AKT and caspase-3 protein expression. A Cell Counting Kit-8 (CCK-8) assay test was conducted to explore the cell viability, and we also studied the apoptosis through western blot assay. A glucocorticoid-induced osteoporosis (GIOP) model was used to confirm the anti-ROS and anti-apoptosis ability of teriparatide.
The CCK-8 assay revealed that Dex reduced the proliferative capability of cells significantly, whereas incubation with teriparatide resulted in a remarkable increase in the proliferation of osteocytes. In addition, teriparatide can rescue the effect of inhibiting cell proliferation due to Dex treatment. Immunofluorescence analysis showed that ROS levels increased in Dex-treated MLO-Y4 cells when compared with control groups. However, the Dex+Teriparatide group showed less ROS when compared with the Dex group. The expression of Sod2 and Cat, two antioxidant enzymes crucial for ROS elimination, was decreased in the Dex group, indicating a defect of the enzymatic antioxidant system. Compared to the Dex group, incubation with teriparatide resulted in a significant decrease in caspase-3 level; when compared with the control group, the caspase-3 level was not significantly different, indicating that teriparatide can rescue apoptosis during Dex exposure. Moreover, teriparatide promotes the expression of AKT, and rescues the apoptosis effect caused by Dex. The results of immunofluorescence also showed that Akt was highly expressed in the teriparatide group when compared with the Dex group. The microstructural parameters Tb.Th, BV/TV, and Tb.N in the methylprednisolone (MPS) group were markedly reduced compared with the control group, but additional treatment with teriparatide could remarkably reverse the methylprednisolone-induced reduction of these parameters. Moreover, the parameter Tb.Sp was significantly increased in the methylprednisolone group compared to the control group, and this increase could be inhibited by teriparatide.
Teriparatide can reduce the cellular ROS level caused by glucocorticoids to facilitate the proliferation of osteocytes through activating the AKT pathway. Meanwhile, the activated AKT can inhibit the activity of proteolytic enzyme caspase-3 and prevent the activation of apoptosis cascade.
通过基于AKT信号通路评估活性氧（ROS）来确定特立帕肽在MLO - Y4细胞系中介导的信号通路，该通路调节骨细胞凋亡。
我们进行了DCFH - DA检测以研究ROS在由地塞米松（Dex）引起的MLO - Y4细胞中的作用。实验分为四组：Dex组、Dex + NAC组、Dex + 特立帕肽组和对照组（未作任何处理）。采用实时逆转录聚合酶链反应检测超氧化物歧化酶2（SOD2）和过氧化氢酶（Cat）mRNA表达。用蛋白质免疫印迹法（WB）检测AKT和半胱天冬酶 - 3蛋白表达。进行细胞计数试剂盒 - 8（CCK - 8）检测以探索细胞活力，并且我们还通过蛋白质免疫印迹法研究细胞凋亡。使用糖皮质激素诱导的骨质疏松（GIOP）模型来证实特立帕肽的抗ROS和抗凋亡能力。
CCK - 8检测显示，Dex显著降低细胞增殖能力，而与特立帕肽孵育导致骨细胞增殖显著增加。此外，特立帕肽可以挽救因Dex处理而抑制细胞增殖的作用。免疫荧光分析表明，与对照组相比，Dex处理的MLO - Y4细胞中ROS水平升高。然而，与Dex组相比，Dex + 特立帕肽组显示出较少的ROS。Dex组中Sod2和Cat这两种对ROS消除至关重要的抗氧化酶的表达降低，表明酶抗氧化系统存在缺陷。与Dex组相比，与特立帕肽孵育导致半胱天冬酶 - 3水平显著降低；与对照组相比，半胱天冬酶 - 3水平无显著差异，表明特立帕肽可以挽救Dex暴露期间的细胞凋亡。此外，特立帕肽促进AKT的表达，并挽救由Dex引起的凋亡效应。免疫荧光结果还显示，与Dex组相比，特立帕肽组中Akt高度表达。与对照组相比，甲基泼尼松龙（MPS）组的微观结构参数骨小梁厚度（Tb.Th）、骨体积分数（BV/TV）和骨小梁数量（Tb.N）显著降低，但额外给予特立帕肽可以显著逆转甲基泼尼松龙引起的这些参数的降低。此外，与对照组相比，甲基泼尼松龙组的骨小梁间距（Tb.Sp）参数显著增加，而特立帕肽可以抑制这种增加。
特立帕肽可以降低糖皮质激素引起的细胞ROS水平，通过激活AKT信号通路促进骨细胞增殖。同时，激活的AKT可以抑制蛋白水解酶半胱天冬酶 - 3的活性并防止凋亡级联反应的激活。
